The present application claims the benefit of priority from PCT Application Serial No. PCT/EP98/03632 (WO 98/58055) filed Jun. 16, 1998, and German Application Serial No. 197 25 803.4 filed Jun. 18, 1997.
The present invention concerns antisense nucleic acids, which are directed against specific sequences of RNA molecules originated from HBV, and vectors and host cells containing these antisense nucleic acids.
Chronic infections with the hepatitis-B virus (HBV) play a central role in the pathogenesis of liver cell carcinoma in humans. Hepadna viruses, of which HBV is one, replicate their DNA genome through reverse transcription of an RNA intermediate, the so-called pregenomic RNA. The application of antisense inhibitors represents, in a particularly specific manner, a therapeutic possibility for HBV infection. The inhibition of the propagation of the HBV can take place with the help of the antisense strategy both through inhibition of reverse transcription, with pregenomic RNA as the target, as well as through blocking of translation, with viral mRNAs as targets. Pregenomic RNA represents the most reasonable RNA target since it is the earliest target for an antisense nucleic acid in the replication cycle of the virus.
In the past, HBV-directed antisense sequences were directed arbitrarily in particular against 5xe2x80x2 regions of viral mRNAs, their cap structure, or the polyadenylation signal (Goodarzi et al., J. Gen. Viro. 71: 3021-3025 (1990); Blum et al., Lancet 337: 1230 (1991); Wu, G. Y. and Wu C. H., J. Biol. Chem. 267: 12436-12439 (1992); Reinis et al., Folia Biol. (Praha) 39: 262-269 (1993); Yao et al., Zhanghua Yi Xue Za Zhi 74: 74-76 (1994); Yao et al., Acta Virol. 39: 227-230 (1995); Yao et al., J. Virol. Hepat. 2: 85-89 (1995); Moriya et al., Biochem. Biophys. Res. Comm. 278: 217-223 (1996)).
Since the selection of the antisense sequences in the past has been rather arbitrary and without rational selection criteria, a large part of the sequences investigated were not effective. Important influence variables on the association behavior between the antisense nucleic acid and the target RNA, such as, for example, the accessibility of the target RNAs to the antisense nucleic acids, were not considered. Furthermore, the structural diversity of the antisense RNAs was not considered and thus the potential of effective antisense inhibitors was not exhausted.
Thus the present invention is based on the object of preparing antisense nucleic acids which hybridize particularly effectively with the pregenomic RNA of human HBV in particular.
This object is realized through the embodiments characterized in the attached patent claims. In particular, an antisense nucleic acid is prepared which is directed against a specific sequence of an RNA molecule originating from HBV as target molecule with the antisense nucleic acid being smaller than the RNA molecule and having an association constant k of at least 5xc3x97103 Mxe2x88x921sxe2x88x921. Preferably the association constant k is at least 2xc3x97104 Mxe2x88x921sxe2x88x921.
The concept xe2x80x9cantisense nucleic acidxe2x80x9d means a native, semi-synthetic, synthetic, or modified nucleic acid molecule of deoxyribonucleotides and/or ribonucleotides and/or modified nucleotides.
The RNA molecule originating from HBV can be pregenomic RNA, pre-C mRNA, pre-S1 mRNA, pre-S2/S mRNA, or the transcript of the X gene. Preferably the RNA molecule is the pregenomic RNA of HBV. Pre-C mRNA is an RNA transcript initiated at the precore initiation codon of the C gene encoding the nucleopsid protein of HBV. Pre-S1 mRNA is an RNA transcript initiated at the pre-S1 initiation codon of the S gene encoding the xe2x80x9cmajorxe2x80x9d envelope protein HbsAg of HBV. Pre-S1 mRNA is an RNA transcript initiated at the second, or S1, initiation codon of the S gene encoding the xe2x80x9cmajorxe2x80x9d envelope protein of HbsAg of HBV. See Fauci et al., ed. Harrison""s Principles of Internal Medicine, McGraw-Hill, 14th Ed. Pp. 1677-1678.
In a preferred embodiment of the present invention, the antisense nucleic acid contains the antisense oligodeoxynucleotide designated with SEQ ID No. 1 to 3. In another preferred embodiment of the present invention, the antisense nucleic acid contains the antisense ribonucleic acid sequences designated with SEQ ID No. 4 through 121 which rapidly hybridize with the pregenomic RNA.
The preferred antisense nucleic acid sequences, designated with SEQ ID No. 1 through 121, hybridize in vitro with above-average rapidity with the pregenomic RNA and are therefore excellent inhibitors of HBV replication in vivo. Surprisingly it was found that these preferred antisense nucleic acids have a selectivity with respect to pregenomic RNA of HBV even if identical target sequence regions occur on the mRNAs of HBV.
A further object of the present invention is a vector which contains the above defined antisense nucleic acid according to the invention or which contains a corresponding DNA sequence complementary to the antisense nucleic acid which following transcription in suitable host cells results in the above-defined antisense nucleic acid according to the invention. The vector according to the invention can preferably contain suitable regulatory elements such as promoters, enhancers, and termination sequences. In an embodiment according to the invention, the vector can be used, for example, for stable integration of the nucleic acid according to the invention into the genetic material of a host cell.
A further object of the present invention is a host cell which contains the antisense nucleic acid or the vector according to the invention. Suitable host cells are, for example, all mammalian cells which carry sequences of hepatitis viruses, preferably human cells which carry hepatitis B virus sequences.
A further object of the present invention is a medication which contains the antisense nucleic acid or the vector according to the invention, possibly in a pharmaceutically acceptable base and/or diluting agent. The medication according to the invention can be used to inhibit or eliminate disease conditions caused by HBV infections through transient or stable integration of the antisense nucleic acid according to the invention by transformation and/or transfection in host cells infected with HBV.